Fig. 8

Bioymifi acts as an inhibitor of VEGFR1–β-catenin interaction and blocks AGE-induced angiogenesis. A The bound conformation of the VEGFR1 isoform5 and β-catenin was predicted by the ZDOCK algorithm. VEGFR1 isoform5 is displayed in yellow, and β-catenin is displayed in green. B Inhibitor binding pockets on the β-catenin protein mode were predicted using MOE-Site Finder plug-ins. C 293T cells were co-transfected with the Flag-tagged β-catenin-overexpression plasmid and HA-tagged VEGFR1 isoform5-overexpression plasmid followed by Bioymifi treatment with different concentrations in the presence of AGEs (100 μg/mL) and then Co-IP assay was performed with HA tag antibody to analyze the interaction between β-catenin and VEGFR1 isoform5. Experiments were repeated three times with similar results and one representative result is shown. D The molecular structure of Bioymifi. E Computational model of the interaction between Bioymifi and the VEGFR1 isoform5-β-catenin complex generated by AutoDock Vina and the key residues for the interaction between Bioymifi and β-catenin. F A 2D docking model showing the interactions between Bioymifi and β-catenin using MOE software. G HUVECs were pretreated with 2 μM Bioymifi for 2 h and then stimulated with AGEs (100 μg/ml) for 1 h and then β-catenin Y142 phosphorylation was measured. n = 6. H-J 293T cells were transfected with TCF/LEF response element (RE) (H), KDR promoter (I) or HDAC9 promoter (J) luciferase constructs followed by AGEs (100 μg/ml) stimulation in the presence or absence of 2 μM Bioymifi. The Dual-Luciferase Reporter Assay was conducted to detected luciferase reporter activities, and luminescence was normalized to Renilla. n = 5. (K-M) HUVECs were treated with AGEs (100 μg/mL) for 24 h in the presence of 2 μM Bioymifi, and then the CCK8 assay (K), Transwell assay (L), and tube formation assay (M) were carried out to evaluate the OD value, migrated cell number and the tube length, branching points, respectively. n = 4 to 5, scale bar indicates 100 or 200 μm. N Aortic rings were treated with or without AGEs (100 μg/mL) for 6 days in the presence of 2 μM Bioymifi and then stained with isolectin B4 (IB4; green). The mean number of sprout (indicated by red dot) from aortic ring was counted. n = 5, scale bar indicates 100 μm. O Schematic diagram illustrating the function of β-catenin in AGEs-induced diabetic angiogenesis. Data are shown as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001